EP0208647A2 - Supports d'immobilisation pour des procédés chimiques et physiques et leur procédé de préparation - Google Patents

Supports d'immobilisation pour des procédés chimiques et physiques et leur procédé de préparation Download PDF

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Publication number
EP0208647A2
EP0208647A2 EP86630112A EP86630112A EP0208647A2 EP 0208647 A2 EP0208647 A2 EP 0208647A2 EP 86630112 A EP86630112 A EP 86630112A EP 86630112 A EP86630112 A EP 86630112A EP 0208647 A2 EP0208647 A2 EP 0208647A2
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Prior art keywords
bone
support
enzyme
oseine
cleaned
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EP86630112A
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German (de)
English (en)
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EP0208647A3 (fr
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Christopher J. Findlay
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Protein Foods Group Inc
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Protein Foods Group Inc
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Publication of EP0208647A2 publication Critical patent/EP0208647A2/fr
Publication of EP0208647A3 publication Critical patent/EP0208647A3/fr
Priority to US07/624,960 priority Critical patent/US5037749A/en
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the present invention is concerned with new immobilisation supports for chemical and physical processes, and methods of making such new supports.
  • Increasingly many modern chemical processes require the employment of immobilisation supports, for example, for the support of catalysts employed in a chemical reaction.
  • An example of physical processes requiring a support are the various types of chromatography in which a column of support material selectively retains compounds from the fluid flowing through it, and subsequently the retained material is removed for assay.
  • the effective support of catalysts has become increasingly important with the employment of catalysts which are cells or bacteria, or complex chemical substances, specific examples the latter being enzymes which are usually complex protein molecules.
  • a support must be provided for the catalyst which will maximize the surface available for catalytic activity, and will also retain the catalyst against physical removal by the flowing reacting materials, so as to provide for fast efficient reaction with minimum loss of the catalyst.
  • Typical supports that have been used hitherto are polymer gels (for example, alginates, agaroses and polyacrylamides) and cell wall material, but these are relatively soft materials that are not able to withstand high pressures and/or high flow rates of the chemical reactants over and/or through them without disruption and physical loss of the catalyst.
  • polymer gels for example, alginates, agaroses and polyacrylamides
  • cell wall material a relatively soft materials that are not able to withstand high pressures and/or high flow rates of the chemical reactants over and/or through them without disruption and physical loss of the catalyst.
  • Another product that has been used for catalyst support is glass beads of controlled pore size, the catalyst being immobilised thereon by charge attraction; it has been found that this type of support has difficulty with fluid suspensions including particles larger than the pore size (e.g. fluid milk) with extensive fouling of the pores.
  • Other inorganic supports include diatomaceous earth, silica, alumina and sand.
  • U.S. Patent No. 4,572,897 descloses carriers for immobilising enzymes in which carrier particles are prepared by binding inert filler particles with a water soluble binder and granulating the mixture.
  • carrier particles are prepared by binding inert filler particles with a water soluble binder and granulating the mixture.
  • inert fillers One of the very many inert fillers suggested is bone meal, although the preferred fillers are diatomaceous earth and cellulose fibre.
  • a new immobilisation support for use in chemical or physical processes consisting of cleaned, finely-divided bird or animal or fish bone.
  • the cleaned finely-divided bone may be provided with a cross-linking agent for the material to be retained on the support.
  • the cross-linking agent is, for example, a bi-functional compound able to attach itself securely to the oseine of the bone and to the material to be retained.
  • a suitable cross-linking agent for enzymatic catalyst support is, for example, glutaraldehyde which provides a reactive aldehyde group for attachment of the supported enzyme.
  • Bone is a unique natural, mechanically-strong, abundant, non-toxic, composite, structural material, whether obtained from birds, animals or fish, consisting of a relatively inert mineral matrix of calcium phosphate, in the form of crystalline hydroxyapatite that is insoluble at physiological pH values, this matrix having a complementary matrix of a relatively stable organic connective collageneous tissue material, particularly the protein oseine, uniformly distributed therethrough.
  • a protein-carbohydrate complex called the ground substance permeates the collagen/bone matrix and surrounds the collagenous fibres.
  • a fourth major constituent is bone-inhabiting cells (osteocytes) each of which occupies its own cavity in the matrix.
  • All bone is somewhat porous and these pores provide a large surface area upon removal of the various types of tissue with which it is associated.
  • the bones of birds are much less dense and much more porous than those of animals, and will provide a corresponding larger surface area.
  • the bones of fish are more porous but more brittle than those of birds and land animals and correspondingly less satisfactory. Since the collagen is distributed uniformly throughout the bone it forms an excellent medium for the immobilisation on the bone of different types of catalysts, particularly catalyst-containing cellular material and proteinaceous catalysts such as enzymes, which can readily be attached by surface adsorbtion, charge attraction, or chemically by use of suitable cross-linking agents.
  • the high collagen content yields a correspondingly relatively high level of residual amino acids, and particularly carboxyl groups, which facilitate the attachment.
  • the bonding is chemical the immobilisation is likely to be more stable than with surface adsorbtion or charge attraction, so that the catalyst is not readily removed by physical action of the reacting solutions.
  • the catalyst is more likely to have sterically available active sites, so that it is more active than when physically immobilised on an inert support, such as a gel or glass beads.
  • the meat processing industries produce bone as a by-product much of which is not easily disposable. Much of the animal bone is cleaned of tissue and then ground to bone meal for use as animal feed or fertiliser.
  • Another source is the poultry processing industry, especially for fast food restaurants, which has as a by-product large quantities of chicken necks and backs. The processing of these necks and backs for mechanical removal of as much as possible of the usable meat has become a standard practice and results in the production of separate meat and bony fractions, the latter consisting of about 60 - 70% by weight of finely-divided bone interspersed with unseparated meat.
  • 4,340,184 poultry parts are fed under pressure into a cylindrical chamber having a circumferential wall constituted by a porous screen of specific slot aperture size.
  • the relatively soft tissue is expelled through the slot apertures while the harder bone and adhering meat is retained and eventually discharged through a bony fraction outlet.
  • the meat will be of particle size less than about 850 microns, while the bone will be of particle size greater than about 850 microns.
  • the bony fraction thus obtained may be cleaned of tissue by washing each volume of bony fraction with two volumes of caustic soda of 1% concentration at 100°C, the mixture being suitably agitated for a period of about 8 minutes and the resulting extract of the soluble proteins removed for further processing to recover the useful protein and fat.
  • Other periods and different concentrations can of course be used. For example, it is found that a fish bony fraction requires a wash of only 2 minutes duration at this temperature, and a longer wash will begin to dissolve the bone, while a lamb fraction requires a longer wash of about 15 minutes and a pork or beef fraction requires a still longer wash of about 20 minutes.
  • the bony fraction may be subjected to a hot water wash prior to the digestion with caustic soda to remove and render fat that is readily removed by such simple treatment, so as to reduce the amount of alkali that is required.
  • the alkali treatment alone does not effect sufficient removal of the unwanted tissue, it may be subjected to prior or post enzymatic treatment to hydrolyse the cellular tissue and render it more readily soluble when washed.
  • the treatment with strong alkali also has the advantage that it sterilises the resultant support material and renders it free of potential reproduction factors such as viruses, bacteria and cells, and also removes potential feed stocks for such factors such as amino acids.
  • Another advantage for some of the immobilization procedures is that the alkaline treatment leaves the support material positively surface charged, so that it is inherently ready to accept and immobilize a negatively charged supported component.
  • the cleaned bone that is thereby obtained from this particular source is already finely-divided and suitable for use immediately as an immobilisation support. In many processes it will be preferred for the support to be of smaller particle size and the bone may be ground to the required size.
  • Processes employing the support in the form of a fluidised bed will require the particle size to be in a specific uniform range, for example 1 to 2 mm, and this can be achieved by grinding and sieving. It may also be preferred for the bone to be of larger particle size, e.g. to pass through a 4 mesh screen, especially when it is required to fill a large reactor. The bone particles from the separation are frequently of about 0.5 cm size, and slivers of up to 4 cm length also occur. Other processes may of course produce finely divided bone of other size ranges and distribution. Bone is a natural, non-toxic, degradable material that is acceptable within quite wide limits as to particle size and volume content as a food constituent, so that it is more widely applicable to food processing systems. Thus, finely divided bone is already approved for use as a source of calcium in various food and vitamin supplements.
  • an assay of chicken bone fraction material from such a mechanical separator showed the following composition, expressed as approximate percentages by weight:
  • the clean bone thus obtained was stored in a 25% brine solution for future use.
  • the liquid fraction obtained from the last-mentioned separation was neutralised with hydrochloric acid and drum dried to obtain about 18 parts of solid material of which 14 parts was protein and 4 parts salt.
  • membrane dialysis could be used to obtain 14 parts of salt-free protein.
  • the solid chicken bone product that results is a coarse porous clean white irregular material, characterized as of plate form.
  • the bone was originally finely-divided in the meat/bone separation process and with the final product it is found that about 40% by weight is retained on a 10 mesh screen (sieve opening 2 mm) while 60% is retained on a 40 mesh screen with some finer particles in the 1 micron size being present; the product may therefore be characterized as being of size such that about 50% by weight is retained by a 20 mesh screen (sieve opening 0.84 mm).
  • the pore size of different bone materials varies widely, as determined by examination with an electron scanning microscope of samples of fish (trout), beef, pork, lamb and chicken vertebrae.
  • the fish bone was found to be very much more porous than any of the other and at low magnification exhibited almost a "honeycomb" structure; the pores were generally large, ovoid in shape with the major axis transverse to the length of the vertebrae, and more uniform in size than in the other bones, varying in the photograph from about 50 to 250 microns along the major axes.
  • the chicken bone was less porous and at low magnification had the appearance of a somewhat porous piece of pumice stone; the high magnification showed pores of from about 100 to 225 microns.
  • the pork bone examined showed areas of large pores adjacent to areas of small pores, the large pores being from about 100 to 220 microns in size while the small pores are about 15 - 35 microns in size.
  • the lamb bone had the external appearance of being very porous with pores of about 100 - 700 microns, but the respective high magnification photograph was of a surface that accidentally was a non-porous surface, so that more accurate measurement of pore size was not possible.
  • the beef bone examined in this manner showed in the low magnification photograph a generally uniform but less porosity than the other bones, the section examined in the high magnification photograph being of highly irregular conformation with apertures from about 15 to 800 microns.
  • Proteinaceous catalyst materials and amino acids to be supported on the bone typically will have molecules of less than 1 micron size, while bacteria and yeasts will typically be of particle size in the range 1 - 5 microns. Clearly therefore these materials can lodge in the apertures in the bone with ready access by the ambient fluid, so that the surface available for attachment is increased enormously by this porosity.
  • the supports of the invention are sterilised, without deactivation of the binding ability of the oseine, the treatment removing unwanted cellular material, bacteria and yeasts from the support, which materials may otherwise start their own fermentations, degradations, etc. Because of the stability of the bone it is also possible to pasteurise it, if necessary, for example by a heat treatment of about 65 - 75°C for a period of about 5 - 30 minutes.
  • This temperature stability of the support also gives the possibility of operating the catalysed process at elevated temperature, for example, at the maximum temperature for a proteinaceous enzymatic catalyst, without degradation of the support.
  • All catalysed systems are temperature sensitive, and the reaction rate of enzymatic systems also increases with temperature up to the temperature (T D ) at which it begins to become denatured.
  • T D temperature at which it begins to become denatured.
  • Oseine is a stable protein which will withstand a higher temperature than moat enzymes.
  • the immobilisation increases the activation energy and renders the immobilised material more stable, there is therefore the possibility that the T D temperature can be shifted into a zone in which pasteurisation occurs and repasteurisation is not needed.
  • Some supported materials are attachable directly to the oseine of the support by adsorbtion, including charge attraction, or by entrapment in the porous material.
  • Direct adsorption to a solid support is one of the preferred methods of immobilization if feasible, because of its simplicity and low cost. Moreover, adsorption is a relatively easily reversible process which allows for ready recovery of the support after the catalyst has been exhausted and involves highly selective binding. In the case of cells this is via multipoint attachment which enables the cells to adhere to the support much more strongly than enzymes.
  • Cell wall composition must be considered, including its charge, the age of the cell, and the ratio between the volume of the cell and its surface area. Additionally, properties of the support such as its composition, its surface charge, surface area and pore size play important roles.
  • the actual charge on the support material limits the available choice of microorganisms for attachment as the adhesion phenomena is mainly based on electrostatic interactions between the charged microbial cells and the charged support. Since electrostatic interactions are involved adsorption will be affected by pH changes that occur as the result of microbial metabolism. All cells that have been examined for attachment, including microorganisms, have a net negative charge.
  • the charge of a cell is related to its surface ionogenic groups, which undergo dissociation according to the pH of the immediate environment, the ionization of carboxyl and amino groups according to Equations 1 and 2 below being apparently a critical reaction indicating a net positivity in highly acidic conditions and a net negativity in alkaline conditions.
  • the p H values for optimum adsorption depend on the relative isoelectric points (iep) of the microbial cells. The strongest adsorption of most cells generally occurs at pH 3 - 6, and the majority of microorganisms studied have iep in the range of p H 2 - pH 3. For example, Leuconostoc mesenteroides has a iep of pH 3.0.
  • the surface charge of a bacterium will be zero while if the pH of a bacterial suspension is above the iep of the carboxyl groups, ionizable hydrogens can be produced, which can conceivably be replaced by any other cation; the entire cell thus behaves as a large anion and is capable of combining with any cation.
  • the bacterial cell can assimilate additional hydrogen ions; each cell will then exhibit a net positive surface charge and behave as a large cation. This charge reversal of some bacteria may not be observed except at extreme pH.
  • the advantage of using a porous support such as bone is related to the amount of surface area available because of this priority.
  • the following is a list of the possible forces of attraction between microbial cells and the adsorbent surfaces:
  • the time required to permit cell adsorption of the cellular material to the solid support must be considered and can be determined by monitoring the optical density of the cell suspension in the feed tank, with respect to time, during recirculation of the cell suspension over the solid support.
  • Maximum cell loading can be considered as having been achieved when the optical density of the cell suspension reaches a constant minimum value, for example, for a period of at least two hours, the recirculation flow rate being maintained at a level that will not cause the cells to be washed off the support.
  • the cell suspension is recirculated for a determined amount of time followed by a period where the solution is allowed to stand in order to encourage maximum adsorption.
  • the recirculating, as with agitation increases the probability of contact between the microbial cells and adsorbent particles, but agitation can not be too long or too vigorous or it can cause desorption.
  • a carefully controlled drying procedure may be used to enhance adsorption by forcing a close contact between the cells and the support surface.
  • Starving the cells e.g. by immersion in pure water
  • Starvation induces a modification of the cell wall and the release of ionic substances, thereby decreasing the electrostatic repulsion between the cells and the support.
  • a decrease, or loss of metabolic activity may be observed following such treatments.
  • the support or the cell surface may be coated with a layer of positively charged colloidal particles such as Al ( OH ) 3 or Fe 2 0 3' or metallic ions (Fe 3+ , Al 3+ ).
  • Adsorbtion may also be used for the attachment of appropriate enzymes and other large-molecule chemical catalysts.
  • Immobilized cells have several advantages over immobilized enzymes, in that it is not necessary to previously have extracted the enzyme from the cell. Furthermore, heat and operational stability in continuous enzyme reactions using intact cells are the same as, or superior to, those of immobilized enzymes.
  • One disadvantage of using immobilized cells is that several different enzymes are usually in the cells and they may initiate side-reactions or degradation of the desired product. This can often be avoided by heat, acid and/or chemical treatment before or after immobilization of microbial cells.
  • Entrapment is based on the inclusion of the supported material within the rigid network constituted by the porous substrate to prevent its diffusion into the surrounding medium, while still allowing penetration of the substrate by the reacting fluid. Within this three dimensional network, the material is free in the compartments and pores.
  • covalent coupling is more appropriately used with dead cells or cells to be utilized for only a single catalytic step.
  • Covalent coupling methods have an advantage over the other methods by reducing or eliminating the problem of release or desorption of cells from the support, and while successfully used for enzyme immobilization, the attachment of whole cells to surfaces requires binding agents which generally are toxic toward the cells. Viable cells immobilized in this manner divide and form new unbound cells, resulting in substantial cell leakage The binding agents also represent an added cost.
  • Some catalytic agents may be attachable directly chemically to the oseine, but it is a relatively stable non-reactive protein and preferably is activated by use of a cross-linking agent, which will attach itself chemically to the oseine and provide a free bond for attachment of the catalyst.
  • a cross-linking agent for use with enzymatic catalysts is glutaraldehyde which will provide a free aldehyde group for chemical attachment of the enzyme cell or biological reagent.
  • glutaraldehyde which will provide a free aldehyde group for chemical attachment of the enzyme cell or biological reagent.
  • the cleaned finely-divided bone is immersed in an aqueous solution of the glutaraldehyde of concentration about 2% by volume for a period of about 10 minutes at a pH in the range 5.5 to 6.5. Concentrations of from 0.1% to 25% can be employed, and p H in the range from 3 to 10.
  • the bone is then water washed two or three times
  • cross-linking agents that have been employed are:
  • Glutaraldehyde has the advantages of it's convenience in use, water solubility, ready availability and relatively very low toxicity.
  • the support of the invention has been employed for the support of the enzymes catalase; B-galactosidase (lactose); pectinases; porcine pepsin; glucose oxidase and glucose isomerase. It is found with some enzymes that account must be taken of the negative ionic effect of the calcium present in the crystalline portion of the bone; for example, pectinase will respond to the available calcium ion and gell, rendering it ineffective for enzymatic action.
  • This effect can be reduced or avoided by "masking" the calcium, for example, by pretreatment with a calcium chelating agent, such as ethyldiaminetetraacetate (EDTA) or alginic acid; or a buffering agent such as sodium citrate and phosphates.
  • a calcium chelating agent such as ethyldiaminetetraacetate (EDTA) or alginic acid
  • EDTA ethyldiaminetetraacetate
  • alginic acid alginic acid
  • a buffering agent such as sodium citrate and phosphates.
  • the use of a buffering agent also provides the possibility of readily controlling the concentration of the enzyme on the support and thus it's specific activity, which can be adjusted to suit the application for which it is employed and perhaps avoid unnecessary provision of the costly material.
  • the bone was treated with the buffer solution in the ratio of 10 mL of buffer per gram of bone, and pectinase enzyme then applied in the concentration of 1 mg per mL of buffer; the resultant activated support showed activity of 10 mg of enzyme per 150 mg of oseine.
  • Lactozym (Trade Mark) 3000 L type HP (lactase) of Novo Industries; and bovine liver catalase
  • the enzyme activity of the invertase was measured by reducing group evolution using 2-cyanoacetamide.
  • Milk clotting activity of the pepsin preparation was measured by timing the initial curd development of reconstituted skim milk (1:10 by volume) in 0.2M acetate buffer at pH 5.8, the activity being expressed as the reciprocal of clotting time in minutes (or milk clotting units) at 25°C.
  • Pectinase activity was measured as with the invertase. Lactase (B-galactosidase) activity was measured using o-nitrophenyl-D - galactopyranoside. The activity of the catalase was measured by the initial rate of oxygen evolution in the presence of 0.5 mM hydrogen peroxide in 0.5 M citrate-phosphate buffer at pH 5.0 using an oxygen polarograph.
  • Enzymatic activity on the bone is expressed in units per gram of dry bone; one unit of activity results in 1 ⁇ mol of substrate at 25°C being reacted per minute.
  • Adsorbtion without any pretreatment was achieved through the addition of the enzyme in an appropriate buffer to the clean dry bone followed by incubation under vacuum for 1 hour and 0°C. Excess enzyme was removed by exhaustive washing with buffer fluid prior to determination of the enzymatic activity. The same procedure for addition to the support was also employed after the respective pretreatment.
  • the respective buffers used were:-
  • the bone was then resuspended in 15ml of solution of 0.05 M acetate buffer and pH 4.4, containing varying amounts of polygalacturonase or invertase. Enzyme coupling proceeded at standard conditions of 0°C for 3 hour under vacuum. The enzyme coupling solution was decanted and the bone thoroughly washed with acetate buffer (0.05 M, pH 4.4) and stored in the acetate buffer.
  • Reagent K 300 mg was added to a suspension of 1 g of bone in 5 ml of 0.1 M sodium phosphate buffer and p H 8.3. The reaction mixture was held under vacuum at room temperature for 1 hour. The solution of Reagent K was withdrawn and the bone rinsed thoroughly with distilled water. The treated bone was immersed with 5 ml of 0.05 M acetate buffer containing varying concentrations of invertase. The mixture was magnetically stirred overnight at 4°C. The enzyme coupling solution was removed and the enzyme-treated bone was washed completely with acetate buffer (pH 4.4, 0.05 M), the bone being stored in 0.05 M acetate buffer at pH 4.4.
  • acetate buffer pH 4.4, 0.05 M
  • cyanamide 100 mg was added to a suspension of 1 g of bone as described above in 5 ml of 0.1 M sodium phosphate buffer of pH 7.0. The mixture was maintained under vacuum for 15-30 min. at room temperature. After extracting the carbodiimide solution, the bone was rinsed with distilled water. The treated bone was placed in 5 ml of 0.05 M sodium acetate buffer at pH 4.4 containing polygalacturonase or invertase at varying concentrations. Enzyme attachment proceeded at standard conditions of 0-4°C for 30 min. under vacuum. The enzyme solution was decanted and the bone washed thoroughly with the acetate buffer and stored in the same buffer.
  • cyanogen bromide 5 g was added to a suspension of 25 g of bone in 200 ml of distilled water. While stirring, 1 M KOH was added dropwise to maintain the pH between 9.5-10.5. After 10 min. the cyanogen bromide solution was withdrawn and the bone washed with sodium bicarbonate at pH 8.0. The bone was immediately resuspended in 25 ml of 0.05 M acetate buffer at pH 4.4, containing polygalacturonase at concentrations used previously. Enzyme coupling proceeded at 0-4°C overnight under vacuum. When immobilization was completed, the treated bone was handled as before.
  • Polygalacturonase activity was measured as follows. To 2.0 ml of 1% (w/v) polygalacturonic acid in 0.05 M acetate buffer (pH 5.0), samples of bone immobilized with enzyme, of decanted enzyme coupling solution or of soluble enzyme possessing polygalacturonase activity were added. The reaction proceeded while stirring for the given reaction period (1-10 min). The reaction mixture (2 ml) was poured into a large test tube containing 10 ml of 0.1 M borate buffer, pH 9.0, to which was added 2 ml of 1% (w/v) 2-cyanoacetamide. Samples were mixed and immersed in a boiling water bath for 10 min.
  • the calibration curve was constructed using solutions of galacturonic acid containing 5-750 nm of galacturonic acid per volume of sample to be assayed.
  • One unit of polygalacturonase was defined was that amount of enzyme required to liberate one micromole of galacturonic acid from the polygalacturonic acid solution at 25°C.
  • Invertase was determined similarly but with some modifications. To 5 ml of 0.05 M sucrose solution in 0.05 M acetate buffer, pH 5.0, a sample of bone immobilized with invertase, of decanted enzyme coupling solution, or of soluble enzyme possessing invertase activity was added. As the reaction progressed with agitation, aliquots of 0.4 ml were removed and added to a test tube that contained 2 ml of 0.1 M borate buffer, pH 9.0. After 0.4 ml of 1% (w/v) 2-cyanoacetamide was added, samples in test tubes were mixed and immersed in a boiling water bath for 10 min. After cooling to 25°C, the absorbance was measured at 276 nanometers.
  • the calibration curve was constructed using equimolar solutions of glucose and fructose containin 5-1000 nanp- mols of each per 0.4 ml.
  • One unit cfinvertase was defined as that amount of enzyme required to liberate one micromole of glucose in one minut for a sucrose solution at 25°C.
  • Table 4 shows the effect of the p H of the coupling solution on the activity of immobilized invertase using hydrazine cross-linking agents.
  • Table 5 shows the effect of the enzyme coupling time on the activity of immobilized invertase using hydrazine.
  • Table 6 shows the effect of invertase concentration in the coupling solution on the activity of immobilized invertase using hydrazine.
  • the effect of the pH of the coupling solution was that the activity of the immobilized invertase decreased as the pH of the enzyme coupling solution increased. This was likely the result of invertase inactivation caused by increases in the pH of the coupling solution during the immobilization as indicated in Table 4.
  • the activity was not affected by coupling time as shown by the results in Table 5. This suggested that for a given enzyme concentration coupling took place immediately when the enzyme was introduced and possible saturation of enzyme coupling sites occurred within 1 and 2.3 hrs respectively using glutaraldehyde and hydrazine respectively.
  • the activity was however affected by enzyme concentration in the coupling mixture.
  • the results obtained reveal that activity of the immobilized invertase is directly related to the amount of available enzyme. It can be seen that approximately 7% of the enzyme was bound in most cases.
  • Silanizing prior to the G HD cross-linking was carried out by treating about 5 g of dry bone for 3 hours at room temperature with a 0.4% solution of 3-aminopropyltriethoxysilane k-APTES). The bone was then rinsed ten times with deionized water, and oven-dried. Immobilization involved the incubation of about 2 g of dry, silanized bone with 10 ml of 2% buffered GHD and pH 5.5). Following rinsing with sodium phosphate buffer at pH 5.5, 10 ml of enzyme solution (2.4 units/ml) were added, and the mixture was allowed to react at 0°C for 90 minutes. After thorough washing with buffer, the level of enzyme activity was determined.
  • the collagenase treatment involved the use of a buffeted 0.2% solution at pH 7.0 of Clostridium histolyticum collagenase, the mixture being reacted overnight at 37° C .
  • the bone was washed free of collagenase using distilled water, and oven-dried prior to treatment with GH D .
  • Ten ml of enzyme solution (2.4 units/ml) were added to about 6 g wet, GHD-activated bone. Coupling proceeded for 90 minutes at 0° C .
  • the enzyme-treated bone was then exhaustively washed with buffer solution.
  • Adsorption involved the incubation of about 2 g dry, untreated bone with 5.0 ml of enzyme solution (2.4 units/ml). Coupling proceeded for 1 to 2 hours at 0° C under vacuum.
  • the immobilization yields in terms of units of enzyme immobilized per gram of support bone (absolute yield), were found to vary widely with the method of immobilization employed.
  • Another application of the material of the invention is in the field of affinity chromatography in which a fluid mixture to be assayed is passed through a column in which specific coupling reactions take place between constituents of the fluid and the material of the column.
  • the usual prior art media for this procedure are various gells which are only capable of slow eleution.
  • the porous bony material of the invention provides for rapid passage of the fluid through the column.
  • When the required couples have been formed the column is washed to remove unwanted material.
  • the wanted couples can then readily be uncoupled by rendering the support sufficiently acid, and washed out from the column.
  • Such procedures are particularly suited for the separation of highly complex and delicate molecules such as antigens.
  • With the supports of the invention it is possible to attach the required coupling agents to the oseine and because of its stable and highly porous nature obtain much faster eleution times.
  • Tests were carried out to compare the pressure drop characteristic of unidirectional fluid flow through a packed bed of the chicken bone support material of invention, as compared with the drop through the same column of 'Dowex' ion-exchange resin (Lot No. MM-12191-A1) manufacturered by Dow Chemical C o., Midland, Michigan.
  • the bed chamber consisted of 2.9 cm inside diameter pipe of methyl methacrylate resin, the total depth of the bed being 19.5 cm with a bed depth of 15.1 cm between upstream and downstream pressure measuring outlets.
  • the pressure differential was measured using a mercury U -tube manometer, while the flow rates were measured by collecting the liquid that passed through the bed in a two litre cylinder, tap water being employed as the liquid and being fed to the packed bed at different flow rates.
  • the bed was packed so that settling was avoided during the tests, and to insure a constant porosity during the test run, an initial flow was maintained at the maximum operating pressure drop to compact the bed until no further change in porosity was detected. The bed was not disturbed until all flow tests were completed.
  • the Reynolds number of the fluid was varied by varying the flow rate.
  • the pressure drops were measured to an accuracy of 0.5 mm of H g, the data being corrected by subtracting the pressure drop in the empty bed and fittings.
  • the equivalent particle diameter of the chicken bone was taken as the average of the opening sizes of 10 and 20 mesh sieves, namely 1.246 mm, its bulk density being 495.6 kg/m 3 .
  • the corresponding equivalent particle size of the resin was 0.635 mm, while its bulk density was 46 2 k g /m 2 .
  • pressure drop through the ion exchange resin is 10 times the pressure drop through the crushed chicken bone.
  • the value A P/V is found to be approximately linear for both the bone and the resin and the consistently higher value for the resin is apparent from the Table.
  • the supports of the invention can of course be employed in any process in which the bone matrix is not appreciably degraded by the conditions of operation, and are particularly applicable to enzyme systems, since the support will usually have much greater tolerance of the operating conditions than will the enzyme itself. Owing to the by-product nature of poultry bone its cost is relatively low and the ease with which enzymes can be immobilised on the protein component renders it highly functional. Examples of suitable applications are:

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
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  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Peptides Or Proteins (AREA)
EP86630112A 1985-07-09 1986-07-08 Supports d'immobilisation pour des procédés chimiques et physiques et leur procédé de préparation Withdrawn EP0208647A3 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US07/624,960 US5037749A (en) 1986-07-08 1990-12-10 Porous immobilization support prepared from animal bone

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US75328685A 1985-07-09 1985-07-09
US753286 1985-07-09

Publications (2)

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EP0208647A2 true EP0208647A2 (fr) 1987-01-14
EP0208647A3 EP0208647A3 (fr) 1989-03-08

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EP (1) EP0208647A3 (fr)
JP (1) JPS6269986A (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100917465B1 (ko) * 2006-08-21 2009-09-14 삼성전자주식회사 비평면 형상의 고체 지지체를 이용하여 미생물을 분리하는방법 및 미생물분리 장치
US7919278B2 (en) 2006-08-21 2011-04-05 Samsung Electronics Co., Ltd. Method of amplifying nucleic acid from a cell using a nonplanar solid substrate
CN106568861A (zh) * 2016-11-04 2017-04-19 辽宁省检验检疫科学技术研究院 鸡肉中氯羟吡啶标准样品、制备方法及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA811104B (en) * 1980-02-26 1982-03-31 Tate & Lyle Ltd Immobilized enzymes, a process for their preparation and their use in converting substrates to products
GB2129809B (en) * 1982-10-06 1986-06-04 Novo Industri As Method for production of an immobilized enzyme preparation by means of a crosslinking agent

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100917465B1 (ko) * 2006-08-21 2009-09-14 삼성전자주식회사 비평면 형상의 고체 지지체를 이용하여 미생물을 분리하는방법 및 미생물분리 장치
US7919278B2 (en) 2006-08-21 2011-04-05 Samsung Electronics Co., Ltd. Method of amplifying nucleic acid from a cell using a nonplanar solid substrate
CN106568861A (zh) * 2016-11-04 2017-04-19 辽宁省检验检疫科学技术研究院 鸡肉中氯羟吡啶标准样品、制备方法及应用

Also Published As

Publication number Publication date
JPS6269986A (ja) 1987-03-31
EP0208647A3 (fr) 1989-03-08
CA1282770C (fr) 1991-04-09

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